Báo cáo y học: " Effector mechanisms of interleukin-17 in collagen-induced arthritis in the absence of interferon-γ and counteraction by interferon-γ" pot

Results Increased levels of IL-17 in CII/CFA-immunized IFN- γR KO mice IFN-γR KO mice develop CIA more readily than wild-type con-trols: symptoms of arthritis usually appear in IFN-γR K

Open Access

Vol 11 No 4

Research article

Effector mechanisms of interleukin-17 in collagen-induced

Hilde Kelchtermans1, Evelien Schurgers1, Lies Geboes1, Tania Mitera1, Jo Van Damme2,

Jacques Van Snick3, Catherine Uyttenhove3 and Patrick Matthys1

1 Laboratories of Immunobiology, Rega Institute, Faculty of Medicine, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

2 Molecular Immunology, Rega Institute, Faculty of Medicine, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

3 Ludwig Institute for Cancer Research, Brussels Branch, Cellular Genetics and Experimental Units, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium

Corresponding author: Patrick Matthys, Patrick.Matthys@rega.kuleuven.be

Received: 3 Mar 2009 Revisions requested: 9 Apr 2009 Revisions received: 29 Jul 2009 Accepted: 17 Aug 2009 Published: 17 Aug 2009

Arthritis Research & Therapy 2009, 11:R122 (doi:10.1186/ar2787)

This article is online at: http://arthritis-research.com/content/11/4/R122

© 2009 Kelchtermans et al.; licensee BioMed Central Ltd

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction Interleukin (IL)-17 is a pro-inflammatory cytokine in

rheumatoid arthritis (RA) and collagen-induced arthritis (CIA)

Since interferon (IFN)-γ inhibits Th17 cell development, IFN-γ

receptor knockout (IFN-γR KO) mice develop CIA more readily

We took advantage of this model to analyse the mechanisms of

action of IL-17 in arthritis The role of IFN-γ on the effector

mechanisms of IL-17 in an in vitro system was also investigated.

Methods IFN-γR KO mice induced for CIA were treated with

anti-IL-17 or control antibody The collagen type II (CII)-specific

humoral and cellular autoimmune responses, myelopoiesis,

osteoclastogenesis, and systemic cytokine production were

determined Mouse embryo fibroblasts (MEF) were stimulated

with IL-17, tumor necrosis factor (TNF)-α and the expression of

cytokines and chemokines were determined

Results A preventive anti-IL-17 antibody treatment inhibited CIA

in IFNγR KO mice In the joints of anti-IL-17-treated mice,

neutrophil influx and bone destruction were absent Treatment

reduced the cellular autoimmune response as well as the

splenic expansion of CD11b+ cells, and production of myelopoietic cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-6 IL-17 and TNF-α synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NFκB ligand (RANKL) in MEF This induction was profoundly inhibited by IFN-γ in a STAT-1 (signal transducer and activator of transcription-1)-dependent way

Conclusions In the absence of IFN-γ, IL-17 mediates its

pro-inflammatory effects mainly through stimulatory effects on

granulopoiesis, neutrophil infiltration and bone destruction In

vitro IFN-γ profoundly inhibits the effector function of IL-17.

Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-γ, this may be an additional mechanism through which IFN-γ attenuates autoimmune diseases

CFA: complete Freund's adjuvant; CHO: Chinese hamster ovary; CIA: collagen-induced arthritis; CII: collagen type II; DTH: delayed type hypersen-sitivity; ELISA: enzyme-linked immunosorbent assay; EMEM: Eagle's minimal essential medium; FCS: fetal calf serum; FITC: fluorescein isothiocy-anate; GCP-2: granulocyte chemotactic protein-2; G-CSF: granulocyte stimulating factor; GM-CSF: granulocyte macrophage colony-stimulating factor; IFN: interferon; IFN-γR KO: interferon-γ receptor knock-out; IL: interleukin; IP-10: interferon-gamma-induced protein; IRF: interferon regulatory factor; ITAC: interferon-inducible T cell alpha chemoattractant; KC: keratinocyte-derived chemokine; MCP: monocyte chemotactic protein; M-CSF: macrophage colony-stimulating factor; MEF: mouse embryo fibroblasts; MIG: monokine induced by gamma interferon; MIP: macrophage inflammatory protein; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phycoerythrin; RA: rheumatoid arthritis; RANKL: receptor activator of nuclear factor-κB ligand; RANTES: Regulated upon Activation, Normal T-cell Expressed, and Secreted; STAT: signal transducer and acti-vator of transcription; TGF-β: transforming growth factor-β; TNF-α: tumor necrosis factor-α.

IL-17 is a pro-inflammatory cytokine produced by activated

CD4+ T cells distinct from Th1 or Th2 cells, designated as

Th17 cells [1-3] IL-17 promotes inflammation by enhancing

production of cytokines such as IL-1β, TNF-α, IL-6 and

recep-tor activarecep-tor of nuclear facrecep-tor-κB ligand (RANKL), as well as

chemokines such as macrophage inflammatory protein

(MIP)-2 and IL-8 [4-6] Factors that promote Th17 cell differentiation

and/or expansion are transforming growth factor (TGF)-β, IL-6

and IL-23 [7] Interferon (IFN)-γ, as a contrast, strongly inhibits

development of Th17 cells both in vitro and in vivo [1,3,8].

Anti-IFN-γ added during in vitro Th17 differentiation causes

increased IL-17 expression, and treated cells display

increased expression of the IL-23 receptor (R) [3] This,

together with the observation that IFN-γ decreases the

expres-sion of IL-23R in IFN-γ-deficient CD4+ T cells differentiated

towards a Th17 phenotype, indicates that IFN-γ is able to

inhibit expression of the IL-23R Additionally, IFN-γ-deficient

mice have increased numbers of IL-17-producing T cells

fol-lowing mycobacterial infection as compared with wild-type

mice, and exogenous IFN-γ reduces the frequency of

IL-17-producing T cells in IFN-γ-deficient mice [9]

There is considerable evidence that IL-17 contributes to the

inflammation associated with rheumatoid arthritis (RA) IL-17

is spontaneously produced by RA synovial membrane cultures

and high levels of IL-17 were detected in the synovial fluid of

patients with RA [10,11] In collagen-induced arthritis (CIA),

an animal model reminiscent in several aspects to RA,

treat-ment with neutralizing anti-IL-17 antibody after the onset of

arthritis reduces joint inflammation, cartilage destruction and

bone erosion [12] Authors proposed that the mechanisms

responsible for slowing the disease are suppression of

pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6, and

elimination of the additive/synergistic effects between IL-17

and these pro-inflammatory cytokines In addition, mice

genet-ically deficient in IL-17 or IL-17R were found to be less

sus-ceptible for induction of CIA [13] In contrast, local IL-17

overexpression accelerates the onset of CIA and aggravates

synovial inflammation [14] Evidence that IFN-γ regulates

sus-ceptibility to CIA through suppression of IL-17 comes from the

observation that mice of the prototypical CIA-susceptible

strain DBA/1 demonstrate a high IL-17 and low IFN-γ cytokine

profile as compared with CIA-resistant C57BL/6 mice [15] In

addition, knocking out the IFN-γ gene renders the C57BL/6

mice susceptible to disease and switched their CD4+ T cell

differentiation towards Th17

Despite the exciting new knowledge about Th17 cells and

IL-17, their mechanisms of action in the pathogenesis of arthritis

are still unclear In the present study we investigated

pro-inflammatory characteristics of IL-17 using the CIA model As

IFN-γ is counteracting the development of Th17 cells, we

chose to induce CIA in IFN-γR knock out (KO) mice Through

neutralization of IL-17 using monoclonal anti-IL-17 antibody,

we tested the effect of endogenous IL-17 on various potential effector targets of CIA, such as autoimmune cellular and humoral responses, production of cytokines and chemokines, stimulation of hematopoiesis and osteoclastogenesis We found a clear-cut inhibition of CIA by treatment with anti-IL-17 antibody and the protection was associated with profound inhibition of myelopoiesis and production of myelopoietic cytokines Extensive myelopoiesis is a well-described phe-nomenom in IFN-γR KO mice challenged with (auto)antigen in complete Freund's adjuvant (CFA; reviewed in [16,17]), so in

an in vitro system using murine embryo fibroblast (MEF) cells

we verified whether IL-17 may directly be involved in the induc-tion of myelopoietic cytokines and/or chemokines and whether IFN-γ may influence this process

Materials and methods

Antibodies and cytokines

Recombinant mouse IFN-γ was derived from the supernatant fluid of Mick cells, a Chinese hamster ovary (CHO) cell line developed in our laboratory [18] IFN-γ was purified by affinity chromatography to a specific activity of 108.5units/mg as described [19]

Anti-IL-17A antibody MM17F3 (IgG1-K) was derived from C57Bl/6 mice immunized with IL-17A-ovalbumin complexes

as described [20] Murine IgG1 monoclonal antibody (9E10),

that recognizes part of the human c-myc protein, and is

pro-duced by the MYC1-9E10.2 (ATCC CRL 1729) hybridoma, was used as control antibody [21]

Mice, induction, evaluation and treatment of CIA

Generation and basic characteristics of the mutant mouse strain (129/Sv/Ev) with a disruption in the gene encoding for the α-chain of the IFN-γ R (IFN-γR KO) have been described [22] These IFN-γR KO mice were backcrossed with wild-type DBA/1 mice for 10 generations to obtain IFN-γR KO DBA/1 mice Homozygous IFN-γR KO mice were identified by PCR as described [23] Wild-type and IFN-γR KO DBA/1 mice were bred in the Experimental Animal Centre of the Rega Institute for Medical Research at Leuven

The generation and basic characterization of IFN-γ-deficient mice of the 129 × BALB/c strain have been described [24] These mice were backcrossed for eight generations to the parental BALB/c strain The signal transducer and activator of transcription (STAT)-1-/- and interferon regulatory factor

(IRF)-1-/- mice, on a C57BL/6 background, were from Dr David E Levy of the New York University School of Medicine (New York, USA) and from Dr Tak Mak of the Ontario Cancer Insti-tute (Ontario, Canada), respectively The generation and char-acteristics of these mice have been described [25,26] C57BL/6 mice (Harlan, Zeist, the Netherlands) were used as wild-type controls

Chicken collagen type II (CII; Sigma-Aldrich, St Louis, MO,

USA) was dissolved at 2 mg/ml in PBS containing 0.1 M

ace-tic acid by stirring overnight at 6°C and emulsified in an equal

volume of CFA (Difco Laboratories, Detroit, MI, USA) with

added heat-killed Mycobacterium butyricum (1.5 mg/ml) Mice

were sensitized with a single intradermal injection at the base

of the tail with 100 μl of the emulsion on day 0, and treated

with 0.2 mg of neutralizing anti-IL-17 or control antibody (in

250 μl PBS) once a week Clinical and histological severity of

arthritis were recorded following a scoring system as

described [27,28]

All animal experiments were approved by the local ethical

com-mittee (University of Leuven)

Measurement of total and anti-CII IgG antibodies and

delayed-type hypersensitivity to CII

Blood samples were taken from the orbital sinus and were

allowed to clot at room temperature for one hour and at 4°C

overnight Individual sera were tested for antibodies directed

to chicken CII by ELISA as described [27] For the

determina-tion of CII-specific IgG1, IgG2a and IgG2b antibody, plates

were incubated for two hours with biotinylated rat antibody to

mouse IgG1, IgG2a or IgG2b (Zymed Laboratories, San

Fran-cisco, CA, USA), followed by a one-hour incubation with

streptavidin-conjugated peroxidase For measurement of total

IgG antibody, plates were coated with goat anti-mouse IgG

(Jackson Immunoresearch Laboratories, West Grove, PA,

USA; 10 μg/ml; 100 μl/well), followed by the same procedure

as described in [27]

For evaluation of delayed-type hypersensitivity (DTH)

reactiv-ity, CII/CFA-immunized mice were subcutaneously injected

with 20 μg of CII/20 μl PBS in the left ear and with 20 μl PBS

in the right ear DTH response was calculated as the

percent-age swelling (the difference between the increase of thickness

of the left ear and the right ear, divided by the thickness of the

right ear, multiplied by 100)

Isolation of splenocytes and mouse embryo fibroblasts

Spleens were harvested, gently cut into small pieces and

passed through cell strainers (Becton Dickinson Labware,

Franklin Lakes, NJ, USA) Red blood cells were lysed by two

consecutive incubations (5 and 3 minutes at 37°C) of the

sus-pension in NH4Cl (0.83% in 0.01 M Tris-HCl, pH 7.2)

Remaining cells were washed and counted

To exclude any interference from endogenous IFN-γ, MEF

were from IFN-γ KO BALB/c origin (unless otherwise

men-tioned) MEF were isolated from mouse embryos between 16

and 18 days of gestation, as described [28] Two times 105

MEF in a total volume of 300 μl Eagle's minimal essential

medium (EMEM) containing 2% heat-inactivated FCS were

seeded in chamber slides (LAB-TEK Brand Products, Nalge

Nunc International, Naperville, IL, USA) After an incubation of

48 hours, cells were stimulated with IL-17 (20 ng/ml) (R&D systems, Abingdon UK) and/or TNF-α (20 ng/ml) (R&D sys-tems, Abingdon UK) in the presence or absence of IFN-γ (100 units/ml) for 48 hours Supernatants were collected and cells were harvested using a cell scraper (LAB-TEK Brand Prod-ucts, Nalge Nunc International, Naperville, IL, USA) Cells were washed and pellets were used for PCR

Flow cytometry

Single-cell splenocyte suspensions (0.5 × 106 cells) were incubated for 15 minutes with the Fc-receptor-blocking anti-bodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA) Cells were washed with PBS (2% FCS) and stained with the indicated fluorescein isothiocy-anate (FITC)-conjugated antibodies (0.5 μg) for 30 minutes, washed and incubated with the indicated phycoerythrin (PE)-conjugated antibodies for 30 minutes FITC-(PE)-conjugated anti-CD25 (7D4) and FITC-conjugated anti-B220 were purchased from BD Biosciences Pharmingen, San Diego, CA, USA) FITC-conjugated CD11b (M1/70), FITC-conjugated anti-CD8 (53-6.7), PE conjugated anti-CD4 (L3T4), and PE-conju-gated anti-Gr-1 (RB6-8C5) were from eBioscience (Immuno-Source, Halle-Zoersel, Belgium) Cells were washed, fixed with 0.37% formaldehyde in PBS and flowcytometric analysis was performed on a FACScan flow cytometer with Cell Quest® software (Becton Dickinson, San Jose, CA, USA)

Detection of cytokines by quantitative PCR and ELISA

MEF were obtained as described above RNA was extracted using the Micro-to-Midi Total RNA Purification System (Invitro-gen Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions cDNA was obtained by reverse transcription using Superscript II Reverse Tran-scriptase and random primers (Invitrogen Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer's instructions For real-time PCR we used a TaqMan® Assays-on-Demand™ Gene expression Product from Applied Biosys-tems (Foster City, CA, USA) Expression levels of granulocyte chemotactic protein-2 (GCP-2) (assay ID Mm00436451_g1, Applied Biosystems, Foster City, CA, USA), IL-6 (assay ID Mm00446190_m1, Applied Biosystems, Foster City, CA, USA), RANKL (assay ID Mm00441908_m1, Applied Biosys-tems, Foster City, CA, USA), IP-10 (assay ID Mm99999072_m1) and granulocyte macrophage colony-stimulating factor (GM-CSF; assay ID Mm99999059_m1) were normalized for 18S RNA (Cat No 4319413E, Applied Biosystems, Foster City, CA, USA) expression Analysis was performed in an ABI Prism 7000 apparatus (Applied Biosys-tems, Foster City, CA, USA) under the following conditions: inactivation of possible contaminating amplicons by AmpEr-ase uracil-N-glycosylAmpEr-ase at 50°C for two minutes, initial dena-turation at 95°C for 10 minutes, followed by 40 thermal cycles

of 15 seconds at 95°C and 90 seconds at 60°C The relative gene expression was assessed using the 2-ΔΔCT method [29]

Expression of cytokines (i.e IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10,

GM-CSF, IFN-γ and TNF-α) was determined by the Bio-Plex

200 system, plex Mouse Cytokine 8-plex assay and

Bio-plex Mouse IL-6 Assay (Bio-Rad, Hercules, CA) IL-17, IP-10

and GM-CSF levels were measured by ELISA (R&D systems,

Abingdon UK) GCP-2 was detected by an ELISA developed

in our laboratory, as described [28]

Results

Increased levels of IL-17 in CII/CFA-immunized IFN- γR

KO mice

IFN-γR KO mice develop CIA more readily than wild-type

con-trols: symptoms of arthritis usually appear in IFN-γR KO mice

from day 16 onwards compared with day 30 in wild-type

ani-mals [30] In a first experiment, we confirmed the inhibitory activity of IFN-γ on the production of IL-17 Thus, IFN-γR KO and wild-type mice were immunized with CII in CFA At day 21,

a time point when the differences in disease symptoms between both groups are most pronounced, mice were injected with anti-CD3 and sera were collected As expected, levels of IL-17 were more than four times higher in the sera of immunized IFN-γR KO mice as compared with those of

wild-type counterparts (Figure 1a, in vivo) Similarly,

anti-CD3-stim-ulated lymphocytes of immunized IFN-γR KO mice produced levels of IL-17 that were more than 10 times higher than those

of wild-type mice (Figure 1a, in vitro) Therefore, to study the

effector functions of IL-17 in arthritis, we chose IFN-γR KO mice for the induction of arthritis

Figure 1

Treatment with anti-IL-17 antibody prevents CIA in IFN-γR KO mice

Treatment with anti-IL-17 antibody prevents CIA in IFN-γR KO mice (a) Interferon-γ receptor knock-out (IFN-γR KO) and wild-type mice were

immu-nized with collagen type II (CII) in complete Freund's adjuvant (in vivo) At day 21 post immunization, five IFN-γR KO and six wild-type mice were

challenged with 10 μg anti-CD3 antibody and serum levels of IL-17 were determined 1.5 hours later Bars represent averages ± standard error of

the mean (SEM) In vitro: lymph nodes were isolated at day 35 post immunization, and lymphocytes were stimulated with 3 μg/ml anti-CD3 antibody Supernatants were collected after 72 hours and tested for IL-17 expression Bars represent averages ± SEM of triplicate cultures * P < 0.05 for

comparison with IFN-γR KO mice (Mann-Whitney U-test) (b, c) Immunized IFN-γR KO mice were injected with anti-IL-17 or control antibody (b) Cumulative incidence of arthritis and (c) mean arthritic score of mice are shown Error bars indicate SEM Data are representative for three

inde-pendent experiments * P < 0.05 (day 19) and 0.005 (from day 20 onwards) for comparison with control-treated mice (Mann-Whitney U-test) (d)

The mean arthritic score on day 21 of arthritic mice only is shown for three experiments Error bars indicate SEM The number of arthritic mice to the total number of mice in each group is shown in brackets.

Neutralization of IL-17 inhibits arthritis development in

IFN- γR KO mice

CII/CFA-immunized IFN-γR KO mice were injected with

neu-tralizing anti-IL-17 or control antibody once a week starting

from day 0 (day of immunization) In control-treated IFN-γR KO

mice, symptoms of arthritis appeared from day 14 and reached

a cumulative incidence of approximately 92% In contrast,

mice treated with anti-IL-17 antibody developed a significantly

less severe form of arthritis with a lower incidence (25%) than

control mice (Figures 1b, c) This protective effect was

con-firmed in two independent experiments When the analysis

was restricted to only arthritic mice, severity of arthritis was still

lower in anti-IL-17-treated mice compared with control-treated

mice (Figure 1d) In one such experiment, mice were sacrificed

on day 25 for histological examination of the joints As evident

from data in Figure 2a, the reduced severity of arthritis in

anti-IL-17-treated mice was associated with inhibition of infiltration

of mono- and polymorphonuclear cells, hyperplasia and

pan-nus formation (measured as the fraction of synovial

inflamma-tory tissue, which has invaded bone tissue and forms bone

erosion) In the control-treated group, several multinucleated

osteoclast-like cells were detected at sites of bone erosion

Osteoclast-like cells and polymorphonuclear cells were

com-pletely absent in sections of anti-IL-17-treated mice Figures

2b and 2c show light microscopy on hematoxylin-stained

sec-tions of both groups of mice

Reduced humoral and cellular auto-immune responses

in IFN- γR KO mice treated with anti-IL-17 antibodies

Pathogenesis of CIA is generally considered to depend in part

on both humoral and cellular immune responses against CII

We wanted to define whether the protection against CIA by anti-IL-17 antibodies results from modulation from either of these Total IgG and anti-CII IgG, IgG1, IgG2a and IgG2b were determined in the sera of anti-IL-17- and control-treated IFN-γR KO mice on day 22 post-immunization No differences

in total IgG serum content were detected in the sera of anti-IL-17- and control-treated animals (Figure 3a) Titers of anti-CII antibodies were found to be reduced in the sera of anti-IL-17-treated mice, although values did not reach statistical signifi-cance (Figure 3b) As to subtypes of anti-CII IgG, no differ-ences in anti-CII IgG1 and IgG2b could be detected However, levels of anti-CII IgG2a were significantly lower in sera of anti-IL-17-treated IFN-γR KO mice as compared with control-treated mice (Figure 3c) DTH was tested on day 25 after immunization by injecting 20 μg of CII in the left ears and vehicle in the right ears Bars in Figure 3d represent the per-centages of swelling in the CII-challenged ears, normalized to the swelling of the PBS-challenged ears A significantly lower DTH response to CII was observed in the anti-IL-17-treated mice as compared with that in control mice Thus, the reduced severity of arthritis in anti-IL-17-treated mice appeared to be associated with reduced cellular immune responsiveness to CII

Figure 2

Absence of infiltration of neutrophils and bone destruction in the joints of anti-IL-17-treated mice

Absence of infiltration of neutrophils and bone destruction in the joints of anti-IL-17-treated mice In experiment 1 of Figure 1d, histological analysis

of the joints was performed On day 25, four mice of each group were sacrificed and sections of the fore limbs, hind limbs and ankles were scored

for three parameters of arthritis following hematoxylin staining (a) Histograms represent averages ± standard error of the mean (SEM) * P < 0.005

for comparison with treated mice (Mann-Whitney U-test) (b, c) Pictures of the hematoxylin-stained paraffin sections of the joints of control-treated (b, a representative mouse) and anti-IL-17-control-treated (c, mice with the most severe symptoms) mice are shown In control-control-treated mice, a severe hyperplasia and infiltration of immunocompetent cells in the synovium and pannus formation that penetrates into the bone can be seen (b1, 2) Detail

of the area indicated by respectively the left and right box in b Note the presence of osteoclast-like multinucleated giant cells (arrows in b1), and the

presence of polymorphonuclear cells in b2 (c1) Detail of the area indicated by the right box in c showing a moderate infiltration of mononuclear cells and hyperplasia Scale bars represent (b, c) 50 μm and (b1, b2 and c1) 500 μm.

Inhibition of arthritis in IFN- γR KO mice is associated

with reduced expansion of CD11b+ cells

The more severe form of arthritis in IFN-γR KO mice as

com-pared with wild-type mice is accompanied with an

extramedul-lary hemopoiesis and expansion of the CD11b+ cell population

[31] These expanding CD11b+ splenocytes, containing

mostly immature mononuclear phagocytes and neutrophils,

can act as a source of osteoclasts and may thus indirectly

account for bone destruction in CIA [23] or may contribute to

neutrophil inflammation in the joints IL-17 is known to

stimu-late granulopoiesis in vivo [32] and to induce the production

of hematopoietic cytokines such as IL-6, IL-8 and G-CSF in

vitro [5], so we analysed the effect of IL-17 neutralization on

the expansion of the CD11b+ cell expansion Therefore,

spleens were isolated from anti-IL-17-treated and control-treated IFN-γR KO mice on day 21 after immunization The mean number of splenocytes was significantly lower in mice treated with anti-IL-17 as compared with control antibody (Fig-ure 4a) To characterize these splenocytes, flowcytometric analysis was performed Figure 4b shows that the anti-IL-17 antibodies inhibit the expansion of the CD11b+ cell popula-tion In fact, treatment with anti-IL-17 antibodies resulted in significantly lower net numbers of CD11b+Gr-1high neutrophils

in the spleen Numbers of CD4+ and CD8+ T cells were also significantly lower in anti-IL-17-treated mice, although to a lesser degree B220+ cell numbers were comparable in both groups of mice These data were confirmed by cytospin splenocyte preparations (Figure 4c) Significantly lower

num-Figure 3

Reduced humoral and cellular immune response following treatment of CII/CFA immunized IFN-γR KO mice with anti-IL-17 antibodies

Reduced humoral and cellular immune response following treatment of CII/CFA immunized IFN-γR KO mice with anti-IL-17 antibodies Interferon-γ receptor knock-out (IFN-γR KO) mice were immunized and injected intraperitoneally with 0.2 mg of neutralizing anti-IL-17 or control antibody once a

week (a, b) On day 22, sera of individual mice were analyzed for (a) total IgG, (b) anti- collagen type II (CII) IgG and (c) anti-CII IgG1, IgG2a and IgG2b Histograms represent averages ± standard error of the mean (SEM) (d) 25 days after immunization, mice in each group were challenged

with 20 μg of CII in the left ear and vehicle in the right ear Delayed type hypersensitivity responses were measured as the percentage of swelling (i.e 100 × the difference between the increase of thickness of the left and the right ears, divided by the thickness of the right ear) at the indicated

time points Histograms represent averages ± SEM * P < 0.05 for comparison with control-treated mice (Mann-Whitney U-test) CFA = complete

Freund's adjuvant.

bers of immature and mature neutrophils were observed in the

spleen of anti-IL-17-treated as compared with control-treated

mice Although to a lesser degree, a significant reduction in

the number of macrophages in the splenocyte population of

17-treated mice was also seen Treatment with

anti-IL-17 antibodies did not significantly affect the number of

lym-phocytes in the splenocyte preparations

Anti-IL-17 treatment in IFN- γR KO mice decreases the

production of hemopoietic cytokines

The reduced expansion of the CD11b+ cell population

follow-ing treatment with anti-IL-17 antibodies might result from

inhi-bition of the production of hemopoietic cytokines Therefore,

sera of CD3-challenged IFN-γR KO mice treated with

anti-IL-17 or control antibodies were collected on day 21 and tested for the presence of cytokines Levels of GM-CSF, IL-6 and IL-12, known to stimulate hemopoiesis, were significantly reduced in the sera of mice treated with anti-IL-17 antibodies

as compared with control-treated mice (Figure 4d), possibly providing an explanation for the reduced expansion of CD11b+ cells in the spleen of anti-IL-17-treated mice Expres-sion of IFN-γ, known to inhibit the CFA-induced expanExpres-sion of CD11b+ cells, was also found to be significantly reduced upon treatment with anti-IL-17 However, as IFN-γR KO mice were used, levels of IFN-γ have no effect in our model With regard

to the expression of other cytokines, levels of the pro-inflam-matory cytokine TNF-α were slightly reduced upon treatment with anti-IL-17 antibodies (1133.3 ± 658.6 and 721.9 ±

Figure 4

Anti-IL-17 antibody treatment reduces the splenic CD11b + cell expansion and the systemic production of myelopoietic cytokines

Anti-IL-17 antibody treatment reduces the splenic CD11b + cell expansion and the systemic production of myelopoietic cytokines Interferon-γ

recep-tor knock-out (IFN-γR KO) mice were immunized and injected intraperitoneally with anti-IL-17 or control antibody once a week (a to c) On day 21, spleens of individual mice were isolated and splenocytes were counted (a) Bars represent the mean number of splenocytes ± standard error of the mean (SEM) (b) Splenocytes were characterized by flow cytometry and the percentage of CD11b+ cells, CD11b + Gr-1 high cells, B220 + cells, CD4 +

cells and CD8 + cells was analyzed Bars represent the mean net numbers of three independent experiments, each consisting of three to four mice

per group ± SEM (c) The percentage of lymphocytes, (immature) neutrophils and macrophages was assessed on cytospin splenocyte preparations

of six anti-IL-17- and six control-treated mice, and the net numbers were calculated Bars represent net numbers ± SEM (d) At day 21 post

immuni-zation, mice were challenged with 10 μg anti-CD3 antibody and serum levels of granulocyte macrophage colony-stimulating factor (GM-CSF), IL-6, IL-12 and IFN-γ were determined 1.5 hours later Bars represent the mean of two independent experiments, each consisting of four to five mice per

group ± SEM * P < 0.05 and ** P < 0.005 for comparison with control-treated mice (Mann-Whitney U-test).

562.3 pg/ml for the control- and anti-IL-17-treated mice,

respectively), and expression of all other tested cytokines

(IL-2, IL-4, IL-5 and IL-10) were comparable in the sera of both

groups of mice (data not shown)

IL-17 induces the production of IL-6, RANKL and GCP-2 in

mouse embryo fibroblasts, and this induction is potently

inhibited by IFN-γ

In a next set of experiments, we verified whether the absence

of bone destruction and reduced influx of neutrophils in the

joints, as well as the reduced production of hematopoietic

cytokines such as IL-6 upon anti-IL-17-antibody treatment, is

indirectly due to the reduced inflammation seen in these mice

or may directly result from neutralization of IL-17 As to the

inhi-bition of bone destruction, we compared the osteoclastogenic

capacity of splenocyte populations of immunized

anti-IL-17-treated and control-anti-IL-17-treated mice Thus, splenocytes from both

groups of mice were stimulated with macrophage

colony-stim-ulating factor (M-CSF) and RANKL for induction of

osteoclas-togenesis We found similar numbers and activity of

osteoclasts in both groups of mice (data not shown),

indicat-ing that osteoclast precursors are present in both splenocyte

populations Possibly, the migration of these osteoclast

pre-cursors to the joints or the production of osteoclast stimulating

factors such as RANKL in the joints is disturbed in

anti-IL-17-treated mice As fibroblasts are an important source of

RANKL, IL-6 and the neutrophil-specific chemokine GCP-2

([28]and our unpublished results), and because of the limited

source of mouse synovial fibroblasts, we chose to use MEF for

in vitro stimulation Cells were stimulated with IL-17 in the

absence or presence of TNF-α for 48 hours, and mRNA levels

were measured using quantitative PCR In addition, we tested

the effect of IFN-γ on the induction of RANKL, IL-6 and

GCP-2 As shown in Figure 5a, IL-17 induced the expression of

GCP-2 mRNA in MEF Moreover, a synergy between IL-17

and TNF-α in the induction of GCP-2 mRNA could be

observed These findings were confirmed at the protein level,

using a GCP-2-specific ELISA (Figure 5b) IL-17 and TNF-α

were also found to synergistically induce the expression of

IL-6 mRNA (Figure 5c), IL-IL-6 protein (Figure 5d) and RANKL

mRNA (Figure 5e) These data indicate that IL-17 is able to

induce the production of neutrophil-specific chemokines such

as GCP-2 Through induction of IL-6 and RANKL, IL-17 can

stimulate hemopoiesis and bone destruction Importantly,

expression of GCP-2, IL-6 and RANKL was counteracted by

IFN-γ (Figures 5a to 5e) Thus, aside from inhibition of the

pro-duction of IL-17, IFN-γ can inhibit the effector function of IL-17

To exclude that IFN-γ non-specifically inhibits cytokine

produc-tion, we also tested the effect of IFN-γ on the production of

keratinocyte-derived chemokine (KC), macrophage

inflamma-tory protein (MIP)-2, IP-10, Regulated upon Activation, Normal

T-cell Expressed, and Secreted (RANTES), monocyte

chemo-tactic protein (MCP)-1, interferon-inducible T cell alpha

chem-oattractant (ITAC), and monokine induced by gamma

interferon (MIG) IFN-γ was found to exhibit stimulatory effects

on the production of MIP-2, IP-10, RANTES, ITAC and MIG induced by IL-17 and/or TNF-α (Figure 5f and data not shown) No significant effect of IFN-γ was observed on the production of M-CSF, KC and MCP-1 (Figure 5f and data not shown)

Inhibition of the effector function of IL-17 by IFN- γ is STAT-1- but not IRF-1-dependent

STAT-1 is a major mediator of cell activation by IFN-γ [33] However, IFN-γ also activates STAT-1-independent pathways, and it has been proposed that these STAT-1-independent pathways mediate suppressive activities of IFN-γ To investi-gate whether suppression of GCP-2, IL-6 and RANKL expres-sion by IFN-γ was STAT-1-dependent, MEF from wild-type and STAT-1-/- mice of the C57BL/6 strain were prepared At the same time, we investigated whether the inhibition by IFN-γ was dependent on IRF-1, a transcription factor acting immediately downstream of STAT-1, by preparing MEF from IRF-1

-/-C57BL/6 mice In each of the MEF, IL-17 and TNF-α synergis-tically induced the expression of GCP-2, RANKL and IL-6 mRNA (data not shown) To evaluate the importance of

STAT-1 and IRF-STAT-1 in the inhibitory action of IFN-γ, the percentage of inhibition of the expression of GCP-2, RANKL and IL-6 mRNA induced by synergistic action of IL-17 and TNF-α was calcu-lated and compared As shown in Figure 6a, the inhibition by IFN-γ was significantly reduced in STAT-1-/-, but unaffected in IRF-1-/- MEF, as compared with wild-type MEF For the inhibi-tion of the expression of GCP-2, these results were confirmed

at the protein level (Figure 6b) These data indicate that the inhibition by IFN-γ is STAT-1-, but not IRF-1-dependent

Discussion

IL-17 plays a key inflammatory role in the propagation of RA and CIA [11,34,35] IL-17 promotes inflammation through enhancing the production of inflammatory cytokines, such as IL-1β, TNF-α and RANKL, as well as neutrophil-specific chem-okines such as MIP-2 and IL-8 [4,5,36] Inhibition of the IFN-γ signaling enhances development of pathogenic Th17 cells that can exacerbate autoimmunity [3,9] IFN-γR KO mice have been found to experience an accelerated and more severe form of CIA [30] Events contributing to this protective effect

of IFN-γ in CIA are inhibition of the CFA-induced myelopoiesis and osteoclastogenesis, inhibition of the production of

GCP-2 and thus neutrophil infiltration, and stimulation of Treg cell activity [23,27,28,31] Recently, IFN-γ was found to regulate susceptibility to arthritis through suppression of IL-17 [15,37] Although Th17 cells and IL-17 have been studied extensively

in previous years, much of their effector functions remain unknown To observe maximal effects of IL-17 neutralization in CIA, we chose to start from IFN-γR KO mice for the induction

of CIA A preventive treatment with the anti-IL-17 antibodies (starting from the day of the immunization), almost completely abrogated arthritis development in IFN-γR KO mice and inhib-ited the influx of immunocompetent cells (predominantly neu-trophils), hyperplasia of the synovial membrane and bone

Figure 5

Synergy of IL-17 and TNF-α in the induction of GCP-2, RANKL and IL-6 and inhibition by IFN-γ

Synergy of IL-17 and TNF-α in the induction of GCP-2, RANKL and IL-6 and inhibition by IFN-γ (a to d) Mouse embryo fibroblasts (MEF) cells of

interferon-γ ligand knock-out (IFN-γ ligand KO) BALB/c mice were grown to confluence and stimulated for 48 hours with IL-17 (20 ng/ml) and/or

TNF-α (20 ng/ml), or were left untreated in the absence or presence of IFN-γ (100 units/ml) (a, c, e and f) cDNA samples were prepared and

sub-jected to quantitative PCR analysis The relative quantity of granulocyte chemotactic protein-2 (GCP-2), receptor activator of nuclear factor-κB lig-and (RANKL), IL-6, IP-10 lig-and granulocyte macrophage colony-stimulating factor (GM-CSF) mRNA in each sample was normalized to the quantity of

18S RNA (b, f) GCP-2, interferon-gamma-induced protein (IP-10) and GM-CSF protein present in the supernatants of stimulated MEF was meas-ured by ELISA (d) IL-6 protein present in the supernatants was quantified by the Bioplex system Results represent the mean of two cultures ± standard error of the mean Results are representative for (a, b) four and (c to f) two independent experiments.

destruction Lubberts and colleagues found that treatment

with anti-IL-17 antibody after the onset of CIA significantly

reduces the severity of CIA in wild-type mice [12] In line with

our results, lower numbers of multinucleated cells were

present in the joints of the anti-IL-17-treated mice as

com-pared with control mice

IL-17 is known to be a potent stimulator of osteoclastogenesis

through induction of RANKL [10,38] Nonetheless, if

spleno-cytes from immunized anti-IL-17-treated and control mice

were stimulated ex vivo with RANKL and M-CSF for induction

of osteoclastogenesis, in the absence or presence of

exoge-nously added neutralizing anti-IL-17 antibody, no differences

could be observed in the numbers or activity of osteoclasts

(data not shown) More recently, however, Sato and

col-leagues have demonstrated that IL-17 has no effect on

osteo-clastogenesis in the RANKL-M-CSF system, but promotes

osteoclast differentiation in the co-culture system through the

induction of RANKL on osteoblastic cells [39]

Joint inflammation in CIA is assumed to depend in part on

col-lagen-specific humoral and cellular immune reactivity

Treat-ment with anti-IL-17 antibody resulted in reduced, although

not significantly, titers of total anti-CII IgG antibodies No

dif-ferences in anti-CII IgG1 and IgG2b could be detected, but

significantly lower levels of anti-CII IgG2a in sera of

anti-IL-17-treated IFN-γR KO mice were established As IgG2a and IgG1

kinetics indirectly reflect Th1/Th2 responses, these data are

suggestive for a lower Th1 response after treatment with

anti-IL-17 antibodies anti-IL-17 was previously shown to be important

in the induction of autoreactive humoral immune responses because a deficiency in this cytokine is associated with a decline in the autoantibody response in CIA and experimental autoimmune encephalomyelitis [13,40] Recently, IL-17 was found to drive autoimmune responses by promoting the forma-tion of spontaneous germinal centers [41] With regard to the cellular immune responses, we found significantly impaired DTH to CII in IFN-γR KO mice treated with anti-IL-17 antibody

In IL-17 KO mice, Nakae and colleagues have established sig-nificantly reduced proliferative responses of lymph node cells against CII in comparison with that of wild-type mice [13] Taken together, these results demonstrate a crucial role for

IL-17 in the activation of CII-specific cellular responses

As we reported earlier [31], the increased severity of CIA in IFN-γR KO mice is associated with an increased CFA-induced extramedullary expansion of immature CD11b+ macrophages and neutrophils In the present study we showed that

anti-IL-17 antibodies inhibit the expansion of this CD11b+ cell popu-lation Characterization of these CD11b+ splenocytes indi-cated a significant reduction in the numbers of (immature) neutrophils The lower numbers of neutrophils present in the spleen may provide an explanation for the reduced influx of neutrophils in the joints of the anti-IL-17-treated mice IL-17 has been found to act as a stimulatory hematopoietic cytokine

by expanding myeloid progenitors and initiating proliferation of mature neutrophils [42] It has been described to induce the secretion of hematopoietic cytokines such as IL-6 and G-CSF

in fibroblasts, through which these stimulated fibroblasts can sustain the proliferation of hematopoietic progenitors and their

Figure 6

Inhibition of effector functions of IL-17 by IFN-γ is STAT-1-dependent

Inhibition of effector functions of IL-17 by IFN-γ is STAT-1-dependent Mouse embryo fibroblasts (MEF) of wild-type, signal transducer and activator

of transcription (STAT)-1 -/- and interferon regulatory factor (IRF)-1 -/- C57BL/6 mice were grown to confluence and stimulated for 48 hours with IL-17

(20 ng/ml) and TNF-α (20 ng/ml), or were left untreated in the absence or presence of IFN-γ (100 units/ml) (a) cDNA samples were prepared and

subjected to quantitative PCR analysis The relative quantity of granulocyte chemotactic protein-2 (GCP-2), receptor activator of nuclear factor-κB

ligand (RANKL) and IL-6 mRNA in each sample was normalized to the quantity of 18S RNA (b) GCP-2 protein present in the supernatants of stim-ulated MEF was measured by ELISA.(a, b) All cultures were performed in duplicate/triplicate in two independent experiments and the percentage of

inhibition was calculated as 100 × (expression in condition without IFN-γ – expression in condition with IFNγ)/expression in condition without IFN-γ)

Results represent the mean of five cultures ± standard error of the mean * P < 0.05 for comparison with corresponding wild-type and, except for

IL-6, IRF-1 -/- condition and ** P < 0.05 for comparison with corresponding wild-type and STAT-1-/- condition.